This Administrative Supplement request is submitted in response to PA-13-041 Collaborative Activities to Promote Metabolomics Research. The intention of the request is to foster a new collaboration that will characterize the metabolomic response to silymarin (SM), an extract of milk thistle seeds, which prevents liver injury and disease progression in many animal models, and also blocks hepatitis C virus (HCV) infection in cell culture. The Supplement will bring together the laboratories of Dr. Stephen J. Polyak, currently funded by R01AT006842 from NCCAM, and Dr. Thomas O. Metz, an established expert in metabolomics, and Dr. Katrina Waters, an expert in bioinformatics. Both Drs. Metzs and Waters laboratories are at Pacific Northwest National Laboratories (PNNL). The overarching hypothesis of the parent grant is that SM-derived natural products interact with mammalian biomolecules in a specific and productive manner to cause changes in signal transduction and gene expression in a cell to protect the liver. To address the hypothesis, we are using two parallel approaches to discover the mechanisms of action of SM. First, we are using microarray analysis to identify the transcriptional changes of liver cell lines and primary hepatocyte cultures treated with SM and SM-derived pure compounds. Second, using photoaffinity click chemistry, we are capturing and validating cellular targets of SM compounds using mass spectrometry. Microarray data indicate that SM induces rapid changes in hepatocellular gene expression within 4-8 hours after exposure. Intriguingly, many of the changes are in genes associated with cell proliferation and metabolism. These and other emerging data suggest that SM is inducing a rapid metabolic reprogramming of cells. Thus, the purpose of our supplement request is to define how SM modulates the metabolome. The extension of our transcriptional and proteomics-based R01 to include metabolomics will allow us to illuminate, to an unprecedented level of detail, exactly how health-promoting natural products such as SM exert their beneficial effects. The novel data emanating from this expanded research project may establish new paradigms for how cells respond to natural products, which may ultimately lead to refinements in natural product treatments and/or drug development opportunities for a multitude of human diseases.